Tetrazine-mediated bioorthogonal prodrug-prodrug activation

Abstract

The selective and biocompatible activation of prodrugs within complex biological systems remains a key challenge in medical chemistry and chemical biology. Herein we report, for the first time, a dual prodrug activation strategy that fully satisfies the principle of bioorthogonality by the symbiotic formation of two active drugs. This dual and traceless prodrug activation strategy takes advantage of the _INVDA chemistry of tetrazines (here a prodrug), generating a pyridazine-based miR21 inhibitor and the anti-cancer drug camptothecin and offers a new concept in prodrug activation.

Fig. 1. (A) _INVDA reaction between a vinyl ether masked drug (inactive) and the tetrazine masked drug (inactive) leads to an active drug pair (a pyridazine and an alcohol). (B) Reaction between the tetrazine prodrug 1 (masked pyridazine-based miR21 inhibitor 2) and the vinyl-O-camptothecin 3 (caged camptothecin 4) showing the dual and traceless prodrug–prodrug generation of 2 and 4. The inhibition of microRNA 21 and topoisomerase would lead to cell death.

Scheme 1. (A) (i) HCl, EtOH/dioxane (1 : 1). (ii) Methyl thiocarbohydrazidium S7, pyridine, DMF. (iii) Amyl nitrite, CH₂Cl₂. (B) (i) NaSCH₃, NEt₃ (ii) 3-nitrophenylboronic acid, Na₂CO₃, Pd(dppf), dioxane/H₂O (4 : 1). (C) (i) 1,2-dibromoethane, NaH, DMF. (ii) PhSeH, CsOH·H₂O. (iii) (1) NaIO₄, NaHCO₃, CH₃OH/H₂O (5 : 1); (2) DIPEA, CH₃CN.

Fig. 2. (A) Reaction between tetrazine 1 and 5′-O-vinyl deoxyuridine 9 (see ESI for HPLC analysis and reaction kinetics). (B) U87-MG, SK-BK3 and PC3 cells incubated with tetrazine 1 (10 μM), 5′-O-vinyl deoxyuridine 9 (20 μM), miR21 inhibitor 2 (10 μM) and tetrazine 1 (10 μM) with 5′-O-vinyl deoxyuridine 9 (20 μM). Cell viability measured after 72 h (MTT assay, n = 3). *** P < 0.001 and ** P < 0.01 by one-way ANOVA with Tukey post-test. No cytotoxicity was observed for 9 up to 20 μM; (C) flow cytometry histograms of Annexin V assay (FITC labelled) with tetrazine 1 (10 μM), miR21 inhibitor 2 (10 μM), 5′-O-vinyl deoxyuridine 9 (20 μM) and tetrazine 1 (10 μM) with 5′-O-vinyl deoxyuridine 9 (20 μM) after 14 h of incubation with SK-BR3.

Fig. 3. (A) (i) Camptothecin 4, vinyl acetate, Na₂CO₃, [Ir(cod)Cl]₂, 1,4-dioxane, 100 °C, 4 h. The reaction between tetrazine 1 and vinyl-O-camptothecin 3 gave >85% conversion (CH₃OH/CH₃CN/H₂O) within 5 days as determined by HPLC. (B) Cell viability of PC3 cells after incubation with vinyl-O-camptothecin 3 (IC₅₀ = 4.64 ± 1.13 μM) and camptothecin 4 (IC₅₀ = 0.15 ± 0.06 μM) for 72 h at 37 °C; insert is non-linear fit used to determine IC₅₀ values (MTT assay, n = 3).

Fig. 4. Cell viability after treatment with tetrazine 1 (10 μM) 95 ± 14%, vinyl-O-camptothecin 3 (0.5 μM) 101 ± 10%, co-treatment of tetrazine 1 (10 μM) and vinyl-O-camptothecin 3 (0.5 μM) 47 ± 8%, camptothecin 4 (0.5 μM) 38 ± 5%, (PC3, MTT-assay, n = 3) *** P < 0.001 by one-way ANOVA with Tukey post-test.